Doxorubicin (FIG. 1) is a broad-spectrum anthracycline, anti-tumor drug used for the treatment of leukemias, lymphomas and solid tumors and is a main-line drug for the treatment of breast cancer. Unfortunately, Doxorubicin exhibits frequent and dose-limiting or even drug-limiting cardiotoxicity. Additionally, most multidrug-resistant tumors and cancer cells display resistance to Doxorubicin. While these undesirable characteristics have limited the clinical usefulness of Doxorubicin, the drug remains one of the oldest and most used anthracycline anti-tumor compounds due to its substantial toxicity to sensitive cancer cells. For this reason, there has been an intensive search for similar anthracycline compounds or derivatives of Doxorubicin having the same or similar anti-tumor activity with greater specificity and/or activity in drug-resistant neoplastic cells.
Research into the mechanism of action of Doxorubicin led to the discovery of the potent formaldehyde Doxorubicin derivative, Doxoform (DoxF, FIG. 1 and U.S. Pat. No. 6,677,309) which cross-links nuclear and mitochondrial DNA and inhibits equally the growth of sensitive and multidrug-resistant cancer cells. DoxF has shown substantial anti-tumor activity (approximately 100-fold above Doxorubicin) that is attributable to the oxazolidine ring formed by the reaction of Doxorubicin with formaldehyde. Additionally, DoxF is no more toxic to cardiomyocytes than Doxorubicin itself. Unfortunately, DoxF is highly susceptible to hydrolysis and therefore, relatively unstable.
The discovery and characterization of the molecular events leading to the cross-linking of DNA by anthracycline drugs, including the induction of formaldehyde synthesis and its role in DNA cross-link formation, led to the synthesis of Daunoform from daunorubicin (FIG. 2), and Epidoxoform (EpiF, FIG. 3) from epidoxorubicin (FIG. 2). Evaluation of these compounds in tissue culture and mice revealed that, although these conjugates have dimeric structures, they function as prodrugs for the monomeric formaldehyde conjugates that cross-link DNA. EpiF differs in structure from DoxF by having the formaldehyde incorporated in 7-membered rings rather than 5-membered rings because of a difference in stereochemistry at the amino sugar. Early on, EpiF was selected as the lead compound because of its stability in water with respect to loss of formaldehyde (half-life 2 hours). While EpiF proved to be more active than epidoxorubicin in cancer cell growth inhibition and in a murine breast tumor model, it was 50-fold less active than DoxF in cancer cell growth inhibition.
The second lead compound, doxsaliform (DoxSF, FIG. 1), contained the formaldehyde as an N-Mannich base with salicylamide and had a monomeric structure rather than a dimeric structure. The N-Mannich base served as a time-release device (half-life 1 hour) for the Doxorubicin-formaldehyde Schiff base (FIG. 3). It also provided functionality for attaching targeting groups that could direct the construct to receptors overexpressed by tumor cells and their associated angiogenesis. DoxSF was also more effective at tumor cell growth inhibition than Doxorubicin but again 50-fold less active than DoxF.
Additional studies have attempted to use methods of targeting prodrugs of Doxorubicin to tumor cells to increase the specificity of these drugs, thereby reducing the non-specific toxicity and related side effects. Antibody Directed Enzyme Prodrug Therapy (ADEPT) and Gene Directed Enzyme Prodrug Therapy (GDEPT) were promising methods for tumor localization of a prodrug-activating enzyme that have been studied over the past six years for anthracycline-based drugs, and particularly Doxorubicin, as Doxorubicin is a widely-used anti-tumor agent that is relatively easy to derivatize. The most effective Doxorubicin prodrugs to utilize this enzyme-activated approach incorporated a peptide or sugar recognized and cleaved by endogenous or non-native-enzymes near the tumor or its supporting vasculature with the goal of reducing the dose-limiting cardiotoxic side effect of Doxorubicin.
Scheeren and coworkers (Mal. Cancer Therap., 1: 901-911, 2002; J. Org. Chem., 2001: 8815-8830, 2001) developed a plasmin-activated tripartate Doxorubicin prodrug, ST-9802, with reduced cardio and systemic toxicity. Plasmin is a protease over-expressed by numerous cancer types and, although it is found in the bloodstream, its activity is inhibited by α2-antiplasmin and α2-macroglobulin. ST-9802 showed no release of Doxorubicin after incubation in bovine serum for 3 days, indicating good plasmin specificity. While the toxicity of ST-9802 was reduced, some efficacy was lost as well and it failed to match the tumor growth inhibition of Doxorubicin in mice bearing human MCF-7 breast tumors even at an equitoxic dose. A variant of ST-9802 with an elongated Katzenellenbogen-type spacer ST-9905 fared better in mouse efficacy experiments, but at best could only match the activity of Doxorubicin (FASEB J., 18: 565-567, 2004).
In 2004 Springer and co-workers (J. Med. Chem., 47: 2651-2658, 2004) reported on a series of nitrogen mustard prodrugs activated by carboxypeptidase G2 (CPG2) produced by Pseudomonas aeruginosa type RS16. These prodrugs were designed to be activated in vivo by the prior administration of a tumor-specific monoclonal antibody conjugated to CPG2. These nitrogen mustard prodrugs have been evaluated using both ADEPT and GDEPT strategies and one of these compounds, ZD2767, showed significant tumor growth inhibition in mice.
In related studies, the capacity of β-lactamase enzymes from Enterobacter species to selectively hydrolyze the β-lactam ring of cephalosporins and penicillins was used to activate a number of cytotoxin-containing prodrugs (Bioorg. Med. Chem. Lett., 3: 323-328, 1993; Cancer Res., 64: 2853-2857, 2004). When used in conjunction with numerous antibody/β-lactamase enzyme conjugates, the Doxorubicin-cephem prodrug achieved higher intratumoral levels of Doxorubicin and exhibited tumor growth inhibition comparable to Doxorubicin.
Although groundbreaking in their day, these prodrug designs failed to address the issue of drug resistance since they relied on Doxorubicin for their cytotoxic effect and several resistance mechanisms for Doxorubicin are known. Since Doxorubicin is a cation at physiological pH, its capacity to diffuse is limited and, therefore, its bystander effect modest. Indeed, none of the Doxorubicin-containing prodrugs described above outperformed Doxorubicin in mouse xenograft assays.
Although ADEPT has advanced significantly, and even landed a few prodrugs in clinical trials, there is still substantial room for improvement. As antibodies are large molecules (approx. 150 kDa) and therefore diffuse very slowly, they are poor targeting agents for many solid tumors. Due to important advances in protein engineering, recombinant fragments that retain most of the antigen affinity and are significantly smaller (approx. 25 kDa) have been made, but these antibodies still need to be humanized to reduce immunogenic response in the host. Immune response to antibody reduces the effectiveness of therapy by removing the antibody-enzyme conjugate from circulation before it reaches the tumor and often limits therapy to a single ADEPT cycle. In addition, humanized antibody development is expensive and time consuming, and constructs containing enzymes often have significantly-reduced enzymatic activity, ands antibody-enzyme conjugates are large molecules that are slow to penetrate tumors, if they do so at all.
Therefore, there is a need for a novel approach to anti-tumor drug targeting that is quick, efficient, and involves a small molecule as a targeting group. Preferably, such a prodrug approach could incorporate the anti-tumor efficacy of doxorubicin while eliminating, or substantially reducing, the associated cardiotoxicity and simultaneously overcoming the doxorubicin drug resistance displayed by many cancer cells.